1. Field of the Invention
The present invention relates to the detection of proteins using synthetic dyes. More specifically, it relates to novel dyes and novel, improved methods of using the dyes for the detection and quantitation of proteins, particularly during electrophoretic separations.
2. Description of the Prior Art
The most powerful and widely used method for the analysis of proteins is the separation of proteins in inert supports by the application of an electric field across the support which causes the proteins in the sample to migrate in a predictable and reproducible manner. See, e.g., Gel Electrophoresis of Proteins: A Practical Approach edited by Hames, B. and Rickwood, D., IRL Press, Washington D.C. (1989). In general, the supports are made from polymers such as polyacrylamide--a polymer of acrylamide and bisacrylamide--or agarose--a polymer of glucose units. Commonly, the separation of the protein sample is performed without the advantage of visualizing the separate components as they fractionate and then, after the separation, the components are detected by staining the constituents by the use of colored dyes. Such dyes include amido black and Coomassie.RTM. dyes, such as Coomassie Brilliant Blue G-250.RTM. dye (Color Index No. 42655) (referred to hereinafter as Coomassie Blue G-250) or Coomassie Brilliant Blue R-250.RTM. dye (Color Index No. 42660) (referred to hereinafter as Coomassie Blue R-250). Such staining agents are needed to detect the proteins, for almost all proteins are transparent in solution, polyacrylamide or agarose gels, or other inert supports and thus cannot be detected visually. The staining of the proteins is normally accomplished by soaking the support containing the separated sample in a solution of the stain, which impregnates the support completely, followed by the removal of the stain from the regions of the support not containing protein by such means as passive diffusion or electrophoresis. The maximal sensitivity for detection of any protein depends on the affinity of the dye for the protein and the extinction coefficient of the dye-protein complex. However, the smallest detectable levels of protein in an inert support, such as a polyacrylamide or agarose gel, is usually determined after the level of dye in the regions of the support not containing protein is reduced to the lowest possible level, thus allowing the largest visual difference between the stained protein and the support.
By far, the most widely used and accepted variants of the electrophoretic fractionation methods described above are separations of sodium dodecyl sulfate (SDS)-coated proteins in a support of polyacrylamide or agarose. Such methodology has been widely used to, among other things, determine the purity of protein samples; monitor the reaction of proteins with various agents; separate protein samples or segments of a single protein for subsequent immunological, radiological or protein-sequence analysis; and compare the relatedness of proteins by various analytical methods. This separation methodology, usually abbreviated SDS-PAGE when polyacrylamide gel electrophoresis has been used, has also been employed for the isolation of small amounts of pure protein for various end uses, such as diagnostic or therapeutic applications. Detection of the separated proteins on a SDS-PAGE or SDS-agarose gel is commonly done by exposing the gel, and concomitantly staining the protein bands, with a dye, such as a Coomassie Blue dye, as indicated above. In fact, Coomassie Blue dyes, which are also known by other names such as Serva Blue, Brilliant Blue, Cyanin, Indocyanin, and Eriosin Brilliant Cyanin, as well as others, are most commonly used due to their ease of use and great sensitivity in the detection of very small amounts of protein. See Wilson, C., Meth. Enzymol. 91, 236-247 (1983).
Practitioners in the art have combined the resolving power of SDS-PAGE and SDS-agarose gel electrophoresis with other techniques, such as isoelectric focusing of proteins in various inert supports, to provide some powerful fractionation techniques. These techniques, usually referred to as two-dimensional gel electrophoresis (2-D gel electrophoresis) techniques, are normally performed by a first fractionation of the sample on a polyacrylamide or agarose support by virtue of the intrinsic charges on the proteins followed by second fractionation of the separated polypeptide chains in a direction perpendicular to the direction of the first fractionation after coating of the protein sample with SDS. Hames, B. and Rickwood, D. supra. Such techniques are claimed to be capable of separating 5,000 to 10,000 protein components simultaneously, thus making them the most powerful separation methods available. Again, detection of the proteins in the fractionated sample is done by staining of the support after the fractionations by a stain such as Coomassie Blue dye.
In certain procedures, care is taken to preserve the intrinsic enzymatic and structural properties of the protein components in the sample while performing PAGE or agarose gel electrophoresis. Such gels, known as native gels, then allow the detection of some of the separated proteins by virtue of their activities (e.g., enzymatic activities).
The amounts of the individual protein constituents present in samples fractionated on SDS-PAGE gels can be determined by measurement of the amount of dye bound to the protein bands by densitometry or, following elution of the bound dye, by direct absorption methods.
However, as powerful as the techniques like those described above are, there are several disadvantages inherent in them that limit their use.
While various staining agents such as Coomassie Blue dyes can be used to stain proteins in SDS-PAGE and native-PAGE gels, the staining process takes considerable time, exposes the sample to undesirable conditions (such as acidic pH or the presence of organic solvents), and has been reported to entrap, "fix", proteins in the support. Zehr, B. et al., "A One-step, Low Background Coomassie Staining Procedure for Polyacrylamide Gels," Analyt. Biochem., 182: 157-159(1989); Wilson, C.,"Staining of Proteins on Gels: Comparisons of Dyes and Procedures," Methods in Enzymology, supra. Some researchers have tried to circumvent these difficulties by adding the stain to the separation matrix prior to the separation; however such procedures result in stain levels present in the support matrix that make detection of low levels of protein impossible due to the color they give the support. Schragger et al., Anal. Biochem. 173, 201-205 (1988). Other scientists have used other chemical reagents such as ones that allow the detection of lipoproteins in gel supports. However, these methods do not render detectable the majority of proteins and thus are of very limited use. What is needed, then, are general stains (i.e., dyes) that can be used to provide detectability to all or almost all of the protein in a support without deeply coloring the support.
While some protein components can be identified in native gels, many proteins cannot be easily detected in this way because presence of the protein in a support matrix complicates or prevents detection via structural, enzymatic or other attributes of proteins. Again, what is needed to improve such methods are dyes that can be used to provide detectability to a protein in such a matrix without relying on the protein's intrinsic structural or enzymatic properties or other intrinsic characteristics that could provide detectability.
While protein can be quantified in gels with great sensitivity by the use of densitometry and related methods, these procedures require that the dye in the gel matrix be substantially completely removed from the areas of the gel not containing protein. This can take a very long time under some circumstances or can require the use of expensive electrical destaining equipment made for this purpose. What is needed are dyes that can be used to quickly and sensitively detect protein in gel supports yet be rapidly removed, or not require removal at all, from the regions of the support not containing protein.
Finally, another drawback with some dyes, including unmodified Coomassie Blue dyes, is the need for very acidic conditions when the dye is used to quantitate protein in solution. Grossberg et al., U.S. Pat. No. 4,219,337; Bradford et al., U.S. Pat. No. 4,023,933. Because some proteins are insoluble under such conditions, they would not be capable of being measured accurately by such methods. Thus, dyes that can be used to quantitate protein under milder pH conditions would be advantageous.
With reference to Coomassie Blue dyes and derivatives thereof, see also Lillie, Conn's Biological Stains, 9th Ed., Williams & Wilkins Co., Baltimore, Md., USA (1977) and Fleming, U.S. Pat. No. 4,966,854.
The aforementioned needs in the art for improved dyes for staining proteins, particularly in applications involving electrophoretic or other separations of proteins through inert supports, are addressed by the present invention. The invention also provides novel and improved methods for detecting proteins.